Fecal microbiota transplantation enhances cell therapy in a rat model of hypoganglionosis by SCFA-induced MEK1/2 signaling pathway.

Hirschsprung disease (HSCR), one of several neurocristopathies in children, is characterized by nerve loss in the large intestine and is mainly treated by surgery, which causes severe complications. Enteric neural crest-derived cell (ENCC) transplantation is a potential therapeutic strategy; however, so far with poor efficacy. Here, we assessed whether and how fecal microbiota transplantation (FMT) could improve ENCC transplantation in a rat model of hypoganglionosis; a condition similar to HSCR, with less intestinal innervation. We found that the hypoganglionosis intestinal microenvironment negatively influenced the ENCC functional phenotype in vitro and in vivo. Combining 16S rDNA sequencing and targeted mass spectrometry revealed microbial dysbiosis and reduced short-chain fatty acid (SCFA) production in the hypoganglionic gut. FMT increased the abundance of Bacteroides and Clostridium, SCFA production, and improved outcomes following ENCC transplantation. SCFAs alone stimulated ENCC proliferation, migration, and supported ENCC transplantation. Transcriptome-wide mRNA sequencing identified MAPK signaling as the top differentially regulated pathway in response to SCFA exposure, and inhibition of MEK1/2 signaling abrogated the SCFA-mediated effects on ENCC. This study demonstrates that FMT improves cell therapy for hypoganglionosis via short-chain fatty acid metabolism-induced MEK1/2 signaling.

Efficient Meta-couplers Squeezing Propagating Light into On-Chip Subwavelength Devices in a Controllable Way.

On-chip photonic systems play crucial roles in nanoscience and nanoapplications, but coupling external light to these subwavelength devices is challenging due to a large mode mismatch. Here, we establish a new scheme for realizing highly miniaturized couplers for efficiently exciting on-chip photonic devices in a controllable way. Relying on both resonant and Pancharatnam-Berry mechanisms, our meta-device can couple circularly polarized light to a surface plasmon, which is then focused into a spot placed with a target on-chip device. We experimentally demonstrate two meta-couplers. The first can excite an on-chip waveguide (with a 0.1λ × 0.2λ cross section) with an absolute efficiency of 51%, while the second can achieve incident spin-selective excitation of a dual-waveguide system. Background-free excitation of a gap-plasmon nanocavity with the local field enhanced by >1000 times is numerically demonstrated. Such a scheme connects efficiently propagating light in free space and localized fields in on-chip devices, being highly favored in many integration-optics applications.

Adjuvant conditioning induces an immunosuppressive milieu that delays alloislet rejection through the expansion of myeloid-derived suppressor cells.

Advances in immunosuppression have been relatively stagnant over the past 2 decades, and transplant recipients continue to experience long-term morbidity associated with immunosuppression regimens. Strategies to reduce or eliminate the dosage of immunosuppression medications are needed. We discovered a novel administration strategy using the classic adjuvant alum to condition murine islet transplant recipients, known as adjuvant conditioning (AC), to expand both polymorphonuclear and monocytic myeloid-derived suppressive cells (MDSCs) in vivo. These AC MDSCs potently suppress T cell proliferation when cultured together in vitro. AC MDSCs also facilitate naïve CD4+ T cells to differentiate into regulatory T cells. In addition, we were able to demonstrate a significant delay in alloislet rejection compared with that by saline-treated control following adjuvant treatment in a MDSC-dependent manner. Furthermore, AC MDSCs produce significantly more interleukin (IL)-10 than saline-treated controls, which we demonstrated to be critical for the increased T cell suppressor function of AC MDSCs as well as the observed protective effect of AC against alloislet rejection. Our data suggest that adjuvant-related therapeutics designed to expand MDSCs could be a useful strategy to prevent transplant rejection and curb the use of toxic immunosuppressive regimens currently used in transplant patients.

High-efficiency plasmonic vortex generation with near-infrared bifunctional metasurfaces.

Plasmonic vortices have shown a wide range of applications in on-chip photonics due to their fascinating properties of the orbital angular momenta (OAM) and phase singularity. However, conventional devices to generate them suffer from issues of low efficiencies and limited functionalities. Here, we establish a systematic scheme to construct high-efficiency bifunctional metasurfaces that can generate two plasmonic vortices exhibiting distinct topological charges, based on a series of reflective meta-atoms exhibiting tailored reflection-phases dictated by both resonant and geometric origins. As a benchmark test, we first construct a meta-coupler with meta-atoms exhibiting geometric phases only, and experimentally demonstrate that it can generate a pre-designed plasmonic vortex at the wavelength of 1064 nm with an efficiency of 27% (56% in simulation). Next, we design/fabricate two bifunctional metasurfaces with meta-atoms integrated with both resonant and geometric phases, and experimentally demonstrate that they can generate divergent (or focused) or convergent (or defocused) plasmonic vortices with district OAM as shined by circularly polarized light with opposite helicity at 1064 nm wavelength. Our work provides an efficient platform to generate plasmonic vortices as desired, which can find many applications in on-chip photonics.

Morroniside protects HT-22 cells against oxygen-glucose deprivation/reperfusion through activating the Nrf2/HO-1 signaling pathway.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a devastating condition that affects neurodevelopment and results in brain injury in infants. Morroniside (MOR), a natural secoiridoid glycoside, has been found to possess neuroprotective effect. However, the effects of MOR on neonatal HIE are unclear. An in vitro HIE model was established in murine hippocampal neurons HT-22 cells using oxygen-glucose deprivation/reoxygenation (OGD/R) stimulation. Our results showed that MOR improved OGD/R-caused cell viability reduction in HT-22 cells. MOR suppressed the production of reactive oxygen species (ROS) and malondialdehyde (MDA) in OGD/R-induced HT-22 cells in a dose-dependent manner. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) were significantly elevated by MOR. Moreover, MOR treatment caused a significant increase in bcl-2 expression, and obvious decreases in the expression levels of bax, cleaved caspase-3, and cleaved caspase-9 expression. Furthermore, MOR significantly upregulated the expression levels of nuclear Nrf2 and HO-1 in OGD/R-treated HT-22 cells. Additionally, knockdown of Nrf2 or HO-1 abrogated the effects of MOR on OGD/R-induced oxidative stress and apoptosis in HT-22 cells. In conclusion, these findings suggested that MOR protects HT-22 cells against OGD/R via regulating the Nrf2/HO-1 signaling pathway.

Enteric mesenchymal cells support the growth of postnatal enteric neural stem cells.

Interplay between embryonic enteric neural stem cells (ENSCs) and enteric mesenchymal cells (EMCs) in the embryonic gut is essential for normal development of the enteric nervous system. Disruption of these interactions underlies the pathogenesis of intestinal aganglionosis in Hirschsprung disease (HSCR). ENSC therapy has been proposed as a possible treatment for HSCR, but whether the survival and development of postnatal-derived ENSCs similarly rely on signals from the mesenchymal environment is unknown and has important implications for developing protocols to expand ENSCs for cell transplantation therapy. Enteric neural crest-derived cells (ENCDCs) and EMCs were cultured from the small intestine of Wnt1-Rosa26-tdTomato mice. EMCs promoted the expansion of ENCDCs 9.5-fold by inducing ENSC properties, including expression of Nes, Sox10, Sox2, and Ngfr. EMCs enhanced the neurosphere-forming ability of ENCDCs, and this persisted after withdrawal of the EMCs. These effects were mediated by paracrine factors and several ligands known to support neural stem cells were identified in EMCs. Using the optimized expansion procedures, neurospheres were generated from small intestine of the Ednrb-/- mouse model of HSCR. These ENSCs had similar proliferative and migratory capacity to Ednrb+/+ ENSCs, albeit neurospheres contained fewer neurons. ENSCs derived from Ednrb-/- mice generated functional neurons with similar calcium responses to Ednrb+/+ ENSCs and survived after transplantation into the aganglionic colon of Ednrb-/- recipients. EMCs act as supporting cells to ENSCs postnatally via an array of synergistically acting paracrine signaling factors. These properties can be leveraged to expand autologous ENSCs from patients with HSCR mutations for therapeutic application.

Computer-aided quantitative MSCT measurements may be useful for congenital lung malformations surgical approach selection.

PURPOSE: To examine the association between the MSCT quantitative measurements of congenital lung malformations (CLM) and the selection of surgical approaches (lobectomy vs. lung-sparing surgery). METHODS: This retrospective study evaluated CLM surgical cases at our institution from 2016 to 2018. MSCT quantitative measurements were generated by a semi-automated approach: the volume of the lesion (Vlesion), the volume of the lesion-involved lobe (Vlobe), the volume of the lesion-involved lung (Vlung) and the volume of the total lung (Vtotal lung). The proportions of Vlesion to Vlobe (Plesion/lobe), Vlesion to Vlung (Plesion/lung), and Vlesion to V total lung (Plesion/total lung) were calculated. We used Logistics regression to examine whether quantitative measurements were associated with the selection of surgical approaches. RESULTS: 151 patients were included (median age at surgery 6 months). 82 patients underwent lung-sparing surgery, and 69 patients underwent lobectomy. Vlesion (OR 1.51, 95% CI 1.09-2.07), Plesion/lobe (OR 1.78, 95% CI 1.16-2.72), Plesion/lung (OR 1.63, 95% CI 1.13-2.35), and Plesion/total lung (OR 1.58, 95% CI 1.12-2.22) were positively associated with the selection of lobectomy. CONCLUSION: The application of quantified MSCT analysis may provide insight into the quantitative characteristics of CLM, which could be potentially useful for surgical approach selection.

Broadband and high-efficiency spin-polarized wave engineering with PB metasurfaces.

Manipulating circularly polarized (CP) light waves at will are highly important for photonic researches and applications. Recently, while Pancharatnam-Berry (PB) metasurfaces have shown unprecedented capabilities to control CP light, meta-devices constructed so far always suffer from the limitations of low-efficiency and narrow bandwidth. Here, we propose a scheme to construct PB metasurfaces with these two issues well addressed. To verify our idea, two PB meta-devices are designed and fabricated for achieving high-efficiency and broadband photonic spin Hall effect and focusing effect, respectively. Experimental results, in good agreement with full wave simulations, demonstrate the desired functionalities with efficiencies reaching 80% within an ultra-wide frequency band (8.2-17.3GHz). The proposed design scheme is generic and can be extended to high-frequency regimes. Our work can stimulate the realizations of high-performance and broadband PB meta-devices with diversified functionalities.

Involvement of the lncRNA AFAP1-AS1/microRNA-195/E2F3 axis in proliferation and migration of enteric neural crest stem cells of Hirschsprung's disease.

NEW FINDINGS: What is the central question of this study? Long non-coding RNAs (lncRNAs) are widely involved in the progression of Hirschsprung's disease (HSCR), but the role of actin filament associated protein 1 antisense RNA1 (AFAP1-AS1), an lncRNA, in HSCR has not been explored before. What is the main finding and its importance? Downregulation of AFAP1-AS1 blocks enteric neural crest stem cell proliferation, differentiation, migration and invasion and promotes the occurrence of HSCR via the miR-195/E2F3 axis, indicating thatAFAP1-AS might be a potential biomarker for HSCR patients. ABSTRACT: Long non-coding RNAs (lncRNAs) are involved in several human disorders. Nevertheless, it remains unclear whether they are implicated in the phenotypes of enteric neural crest stem cells (ENCSCs) in Hirschsprung's disease (HSCR). Therefore, we designed this study to explore the pathogenicity of AFAP1-AS1 for HSCR. Microarray analysis and bioinformatic tools were used to screen out the differentially lncRNAs and microRNAs (miRNAs) in patients with HSCR. Small interference RNA transfection was applied to carry out functional experiments in ENCSCs. Cellular activities were detected by cell counting kit-8, 5-ethynyl-2'-deoxyuridine, Transwell assays and flow cytometry. Finally, rescue experiments were performed to examine the cofunction of AFAP1-AS1 and miR-195 and of miR-195 and E2F transcription factor 3 (E2F3). AFAP1-AS1 was reduced in HSCR patients. Meanwhile, knockdown of AFAP1-AS1 reduced the cell migratory and proliferative capacities and facilitated cell apoptosis along with G0/G1 phase arrest. E2F3 was diminished when miR-195 was upregulated, and AFAP1-AS1 inhibition reduced its ability to bind to miR-195. Altogether, AFAP1-AS1 silencing acts as an endogenous RNA by interacting with miR-195 to alter E2F3 expression, thus conferring repressive effects on ENCSC activity and promoting HSCR progression.

High-efficiency broadband vortex beam generator based on transmissive metasurface.

Vortex beam has attracted growing attention in recent years due to its remarkable abilities in the communication system since it is believed to be an effective way to improve the channel capacity efficiency. However, available vortex beam generators suffer from the issues of complex configurations, low efficiency as well as narrow bandwidths, especially for the transmissive case. Here, we proposed a broadband transmissive metasurface to generate vortex beam with l=1 in a broad band ranging from 8GHz to 13 GHz. We enhance the working bandwidth by carefully designing the meta-atoms which provide high transmittances along with similar slopes of the phase responses within a large frequency interval. More importantly, the designed vortex beam generator exhibits very high working efficiencies (more than 83% within the whole working band). Our finding opens a door for the design of high-efficiency broadband transmissive vortex beam generators and operating at other frequency domains.

Correlation of spatio-temporal characteristics of intestinal inflammation with IL-17 in a rat model of hypoganglionosis.

Interleukin 17 expression is increased in children with Hirschsprung disease, which is characterized by intestinal inflammation. This study designed to exploit the characteristics of intestinal inflammation and examine the correlation of interleukin 17 in this process of hypoganglionosis model established by benzalkonium chloride treatment. Colon sections from female rats were treated with benzalkonium chloride to induce hypoganglionosis or with saline alone as a sham control. C-reactive protein and tumor necrosis factor-ɑ were used as markers of inflammation. Expression of C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 was assessed in colon tissue and blood serum on days 7, 14 and 21 after treatment. The correlation between C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 expression was estimated using the Spearman's rank-correlation coefficient. C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 were strongly expressed in submucosa and mucosa layers and serum from treated animals. The expression of C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 maintained the highest level at Day 21. Only C-reactive protein and tumor necrosis factor-ɑ expression was increased in control animals and only on day 7. Spearman's rank correlation coefficient was significant in C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 at Day 7, 14 and 21. Concomitant upregulation of C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 and significant positive correlations between C-reactive protein, tumor necrosis factor-ɑ, and interleukin 17 may imply that interleukin 17 is involved in spatio-temporal inflammation induced by benzalkonium chloride.

Trifunctional metasurfaces: concept and characterizations.

Achieving multiple diversified functionalities in a single flat device is crucial for electromagnetic (EM) integration. While many recent efforts were devoted to designing multifunctional metasurfaces, most meta-devices realized so far typically exhibit only two functionalities. In this paper, we propose a generic strategy to design trifunctional metasurfaces, based on carefully designed single structure meta-atoms possessing polarization-controlled transmission/reflection properties. As a proof of our concept, we design and fabricate a trifunctional metasurface possessing simultaneously three distinct functionalities including beam splitting, deflecting, and focusing, and perform both far-field and near-field microwave experiments to demonstrate the predicted functionalities of the fabricated device. Experimental results are in good agreement with numerical simulations. These findings can motivate the realizations of high-performance multifunctional meta-devices in different frequency domains and with diversified functionalities.

Molecular mechanism of estrogen-mediated neuroprotection in the relief of brain ischemic injury.

BACKGROUND: This study aimed to explore the molecular mechanism of estrogen-mediated neuroprotection in the relief of cerebral ischemic injury. The gene expression profiles were downloaded from Gene Expression Omnibus database, and differentially expressed genes (DEGs) were identified using limma package in R software. Further, DEGs were subjected to Gene Ontology (GO) cluster analysis using online Gene Ontology Enrichment Analysis Software Toolkit and to GO functional enrichment analysis using DAVID software. Using the Gene Set Analysis Toolkit V2, enrichment analysis of Kyoto Encyclopedia of Genes and Genomes pathways was performed. In addition, protein-protein interaction (PPI) network was constructed using STRING database, and submodule analysis of PPI network. Lastly, the significant potential target sites of microRNAs (miRNAs) were predicted using Molecular Signatures Database, and the function analysis of targets of predicted miRNA was also performed using DAVID software. RESULTS: In total, 321 DEGs were screened in the estrogen-treated sample. The DEGs were mainly associated with intracellular signaling and metabolic pathways, such as calcium channel, calcineurin complex, insulin secretion, low-density lipoprotein reconstruction, and starch or sugar metabolism. In addition, GO enrichment analysis indicated an altered expression of the genes related to starch and sucrose metabolism, retinol metabolism, anti-apoptosis (eg., BDNF and ADAM17) and response to endogenous stimulus. The constructed PPI network comprised of 243 nodes and 590 interaction pairs, and four submodules were obtained from PPI network. Among the module d, four glutamate receptors as Gria4, Gria3, Grin3a and Grik4 were highlighted. Further, 5 novel potential regulatory miRNAs were also predicted. MIR-338 and MIR520D were closely associated with cell cycle, while the targets of MIR-376A and MIR-376B were only involved in cell soma. CONCLUSIONS: The DEGs in estrogen-treated samples are closely associated with calcium channel, glutamate induced excitotoxicity and anti-apoptotic activity. In addition, some functionally significant DEGs such as BDNF, ADAM17, Gria4, Gria3, Grin3a, Grik4, Gys2 and Ugtla2, and new miRNAs like MIR-338 and MIR-376A were identified, which may serve as potential therapeutic targets for the effective treatment of cerebral ischemic injury.

Combination of exogenous cell transplantation and 5-HT4 receptor agonism induce endogenous enteric neural crest-derived cells in a rat hypoganglionosis model.

Enteric neural crest-derived cells (ENCCs) can migrate into endogenous ganglia and differentiate into progeny cells, and have even partially rescued bowel function; however, poor reliability and limited functional recovery after ENCC transplantation have yet to be addressed. Here, we investigated the induction of endogenous ENCCs by combining exogenous ENCC transplantation with a 5-HT4 receptor agonist mosapride in a rat model of hypoganglionosis, established by benzalkonium chloride treatment. ENCCs, isolated from the gut of newborn rats, were labeled with a lentiviral eGFP reporter. ENCCs and rats were treated with the 5-HT4 receptor agonist/antagonist. The labeled ENCCs were then transplanted into the muscular layer of benzalkonium chloride-treated colons. At given days post-intervention, colonic tissue samples were removed for histological analysis. ENCCs and neurons were detected by eGFP expression and immunoreactivity to p75NTR and peripherin, respectively. eGFP-positive ENCCs and neurons could survive and maintain levels of fluorescence after transplantation. With longer times post-intervention, the number of peripherin-positive cells gradually increased in all groups. Significantly more peripherin-positive cells were found following ENCCs plus mosapride treatment, compared with the other groups. These results show that exogenous ENCCs combined with the 5-HT4 receptor agonist effectively induced endogenous ENCCs proliferation and differentiation in a rat hypoganglionosis model.

Decreased proliferative, migrative and neuro-differentiative potential of postnatal rat enteric neural crest-derived cells during culture in vitro.

A growing body of evidence supports the potential use of enteric neural crest-derived cells (ENCCs) as a cell replacement therapy for Hirschsprung's disease. Based on previous observations of robust propagation of primary ENCCs, as opposed to their progeny, it is suggested that their therapeutic potential after in vitro expansion may be restricted. We therefore examined the growth and differentiation activities and phenotypic characteristics of continuous ENCC cultures. ENCCs were isolated from the intestines of postnatal rats and were identified using an immunocytochemical approach. During continuous ENCC culture expansion, proliferation, migration, apoptosis, and differentiation potentials were monitored. The Cell Counting Kit-8 was used for assessment of ENCC vitality, Transwell inserts for cell migration, immunocytochemistry for cell counts and identification, and flow cytometry for apoptosis. Over six continuous generations, ENCC proliferation potency was reduced and with prolonged culture, the ratio of migratory ENCCs was decreased. The percentage of apoptosis showed an upward trend with prolonged intragenerational culture, but showed a downward trend with prolonged culture of combined generations. Furthermore, the percentage of peripherin(+) cells decreased whilst the percentage of GFAP(+) cells increased with age. The results demonstrated that alterations in ENCC growth characteristics occur with increased culture time, which may partially account for the poor results of proposed cell therapies.

A Time-Limited and Partially Reversible Model of Hypoganglionosis Induced by Benzalkonium Chloride Treatment.

Serosal application of benzalkonium chloride (BAC) has been previously applied to produce a model of aganglionosis; however, confusion remains regarding the extent of chemical ablation of enteric myenteric plexus after BAC treatment. The time sequence of BAC-induced effects on the myenteric plexus of the rat colon was determined and followed the morphologic changes. After sacrifice of animals 7, 14, 28, 56, 84 or 168 days postintervention, colonic tissue samples were removed, fixed in formalin, and cut into 5-µm longitudinal sections for histological analysis. The neural analysis was used to re-evaluate BAC treatments for the appropriate model. Compared with rats in sham groups, rats in 0.1 %-30-min BAC group maintained only 15.27 ± 4.80 % of ganglia per section in a 1-cm/5-µm slice and 11.76 ± 2.30 % of ganglionic cells after 28 days, the lower and stable number of ganglionic cells between Day 7 and 84 (from 11.67 ± 2.10 to 19.05 ± 5.10 %). Although an increase, ganglionic cell numbers did not recover at Day168 when compared with the numbers in sham groups. The results showed that characteristics of rats in the 0.1 %-30-min BAC group between Day 7 and 84 most closely kept in stable state, suggesting that these treatment parameters are ideal for producing a hypoganglia model of hypoganglionosis.

Propranolol induces regression of hemangioma cells via the down-regulation of the PI3K/Akt/eNOS/VEGF pathway.

BACKGROUND: Infantile hemangioma (IH) is a benign vascular neoplasm resulting from the abnormal proliferation of endothelial cells and pericytes in infants. Propranolol, a non-selective ß-adrenergic blocker, has recently emerged as an effective therapy for IH, causing regression. However, its potential therapeutic mechanism remains largely unknown. PROCEDURE: An XPTS-1 cell line was established by isolating hemangioma-derived endothelial cells (HemECs) from a specimen of human proliferating IH. Flow cytometer assay was performed to assess the effect of propranolol on cell cycle distribution. Western blot was employed to determine changes of protein expression. Matrigel invasion and tube formation assays were used to measure invasion ability and tube formation ability, respectively. Commercial kits were employed to quantify NO and VEGF levels. RESULTS: Propranolol blocked norepinephrine-induced HemECs cell cycle progression as well as the expression of cyclin A2 and cyclin D2; whereas p21 and p27 proteins were altered conversely. Propranolol inhibited norepinephrine-induced cell invasion by reducing the expression of MMP-9, VEGF, and p-cofilin. NO and VEGF release induced by norepinephrine was decreased by propranolol pretreatment, coincident with alterations in the phosphorylation of Akt, eNOS, and VEGFR-2. Tube formation ability and subsequent levels of NO and VEGF elevated by norepinephrine were distinctively counteracted in HemECs. CONCLUSIONS: The current study demonstrated the antiangiogenic properties of propranolol in vitro and that the drug was able to induce the regression of hemangioma cells via the inhibition of cell cycle progression, invasion, and tube formation, concomitantly with decreased NO and VEGF levels through the down-regulation of the PI3K/Akt/eNOS/VEGF pathway.

Optogenetic Activation of Cholinergic Enteric Neurons Reduces Inflammation in Experimental Colitis.

BACKGROUND & AIMS: Intestinal inflammation is associated with loss of enteric cholinergic neurons. Given the systemic anti-inflammatory role of cholinergic innervation, we hypothesized that enteric cholinergic neurons similarly possess anti-inflammatory properties and may represent a novel target to treat inflammatory bowel disease. METHODS: Mice were fed 2.5% dextran sodium sulfate (DSS) for 7 days to induce colitis. Cholinergic enteric neurons, which express choline acetyltransferase (ChAT), were focally ablated in the midcolon of ChAT::Cre;R26-iDTR mice by local injection of diphtheria toxin before colitis induction. Activation of enteric cholinergic neurons was achieved using ChAT::Cre;R26-ChR2 mice, in which ChAT+ neurons express channelrhodopsin-2, with daily blue light stimulation delivered via an intracolonic probe during the 7 days of DSS treatment. Colitis severity, ENS structure, and smooth muscle contractility were assessed by histology, immunohistochemistry, quantitative polymerase chain reaction, organ bath, and electromyography. In vitro studies assessed the anti-inflammatory role of enteric cholinergic neurons on cultured muscularis macrophages. RESULTS: Ablation of ChAT+ neurons in DSS-treated mice exacerbated colitis, as measured by weight loss, colon shortening, histologic inflammation, and CD45+ cell infiltration, and led to colonic dysmotility. Conversely, optogenetic activation of enteric cholinergic neurons improved colitis, preserved smooth muscle contractility, protected against loss of cholinergic neurons, and reduced proinflammatory cytokine production. Both acetylcholine and optogenetic cholinergic neuron activation in vitro reduced proinflammatory cytokine expression in lipopolysaccharide-stimulated muscularis macrophages. CONCLUSIONS: These findings show that enteric cholinergic neurons have an anti-inflammatory role in the colon and should be explored as a potential inflammatory bowel disease treatment.

Autologous cell transplantation for treatment of colorectal aganglionosis in mice.

Neurointestinal diseases cause significant morbidity and effective treatments are lacking. This study aimes to test the feasibility of transplanting autologous enteric neural stem cells (ENSCs) to rescue the enteric nervous system (ENS) in a model of colonic aganglionosis. ENSCs are isolated from a segment of small intestine from Wnt1::Cre;R26iDTR mice in which focal colonic aganglionosis is simultaneously created by diphtheria toxin injection. Autologous ENSCs are isolated, expanded, labeled with lentiviral-GFP, and transplanted into the aganglionic segment in vivo. ENSCs differentiate into neurons and glia, cluster to form neo-ganglia, and restore colonic contractile activity as shown by electrical field stimulation and optogenetics. Using a non-lethal model of colonic aganglionosis, our results demonstrate the potential of autologous ENSC therapy to improve functional outcomes in neurointestinal disease, laying the groundwork for clinical application of this regenerative cell-based approach.

Association between gut microbiota and Hirschsprung disease: a bidirectional two-sample Mendelian randomization study.

Background: Several studies have pointed to the critical role of gut microbiota (GM) and their metabolites in Hirschsprung disease (HSCR) pathogenesis. However, the detailed causal relationship between GM and HSCR remains unknown. Methods: In this study, we used two-sample Mendelian randomization (MR) analysis to investigate the causal relationship between GM and HSCR, based on the MiBioGen Consortium's genome-wide association study (GWAS) and the GWAS Catalog's HSCR data. Reverse MR analysis was performed subsequently, and the sensitivity analysis, Cochran's Q-test, MR pleiotropy residual sum, outlier (MR-PRESSO), and the MR-Egger intercept were used to analyze heterogeneity or horizontal pleiotropy. 16S rDNA sequencing and targeted mass spectrometry were developed for initial validation. Results: In the forward MR analysis, inverse-variance weighted (IVW) estimates suggested that Eggerthella (OR: 2.66, 95%CI: 1.23-5.74, p = 0.01) was a risk factor for HSCR, while Peptococcus (OR: 0.37, 95%CI: 0.18-0.73, p = 0.004), Ruminococcus2 (OR: 0.32, 95%CI: 0.11-0.91, p = 0.03), Clostridiaceae1 (OR: 0.22, 95%CI: 0.06-0.78, p = 0.02), Mollicutes RF9 (OR: 0.27, 95%CI: 0.09-0.8, p = 0.02), Ruminococcaceae (OR: 0.16, 95%CI: 0.04-0.66, p = 0.01), and Paraprevotella (OR: 0.45, 95%CI: 0.21-0.98, p = 0.04) were protective factors for HSCR, which had no heterogeneity or horizontal pleiotropy. However, reverse MR analysis showed that HSCR (OR: 1.02, 95%CI: 1-1.03, p = 0.049) is the risk factor for Eggerthella. Furthermore, some of the above microbiota and short-chain fatty acids (SCFAs) were altered in HSCR, showing a correlation. Conclusion: Our analysis established the relationship between specific GM and HSCR, identifying specific bacteria as protective or risk factors. Significant microbiota and SCFAs were altered in HSCR, underlining the importance of further study and providing new insights into the pathogenesis and treatment.